Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Block by 5-hydroxytryptamine of neuronal acetylcholine receptor channels expressed inXenopus oocytes

Summary 1. Effects of 5-hydroxytryptamine (5-HT) on neuronal nicotinic acetylcholine (ACh) receptor channels were investigated by expressing cloned channel subunits inXenopus oocytes.2. When channels were expressed with a combination of ₃ and ₄ subunits, 5-HT (10 to 300 µM) reversibly inhibited an inward current activated by 100 µM ACh in a concentration-dependent manner. The inhibition was also observed when ₃ subunit was combined with ₂ subunit instead of ₄ subunit, or ₄ subunit was combined with ₂ or _4–1 subunit instead of ₃ subunit to express channels.3. Compounds known to antagonize at 5-HT receptors (LY53857, metoclopramide and propranolol) exhibited an agonistic effect: they inhibited the ACh-activated current.4. The results suggest that 5-HT inhibits recombinant neuronal nicotinic receptor channels through a binding-site distinct from conventional 5-HT receptors. The binding-site may not be attributed to a unique type of channel subunits.     

Integrin activation is required for VEGF and FGF receptor protein presence on human microvascular endothelial cells

Endothelial cell proliferation and migration is initiated by growth factors including FGF and VEGF that bind to specific transmembrane receptor tyrosine kinases. Mechanisms that regulate in vivo expression of fibroblast growth factor receptors (FGFR) and vascular endothelial growth factor receptors (VEGFR) are not well understood. Since it is well known that different matrices influence the proliferation and migration of endothelial cells in culture, we hypothesized that changes in the extracellular matrix environment can regulate growth factor receptors on endothelial cells. We cultured human microvascular endothelial cells on different matrices (vitronectin, laminin, fibronectin, fibrin, and collagen IV) and examined for the presence of growth factor receptors (FGFR-1, FGFR-2, VEGFR-1, and VEGFR-2). We show that vitronectin increased the presence of all four growth factor receptors and most notably, VEGFR-1. In contrast, fibrin decreased all four receptors, especially FGFR-1 and FGFR-2. Inhibiting phosphotyrosine signaling abolished immunostaining for all four receptors, regardless of the matrix, but was not dependent on activating the Fyn-Shc pathway. Cells plated on vitronectin in the presence of blocking antibodies to integrins _v3 and _v5 similarly decreased presence of these growth factor receptors. Our data suggests a possible mechanism of how matrix-integrin interactions regulate endothelial cell responsiveness to growth factors and anchorage-dependent cell growth.     

Dipole moments of scorpion toxins direct the interaction towards small- or large-conductance Ca2+-activated K+ channels

Ca²⁺-activated K⁺ channels consist of alarge family of membrane proteins, among which twogroups have been characterized by electrophysiologicalcriteria, the small conductance (SK) and the largeconductance (BK) Ca²⁺-activated K⁺channels. Scorpion toxins that block K⁺ channelsexhibit a common three-dimensional structureconstituted of a short -helix connected bydisulfide bonds to a -sheet. The leiurotoxin I(LTX1) related toxins interact specifically with theSK channel via basic residues of their -helix,while the charybdotoxin (ChTX) family recognizes theBK channel with basic residues of their -sheet.In an attempt to better understand thestructure–activity relationships of these toxins andthe characteristics of the electrostatic interactionswith the receptor site, we investigated theelectrostatic potential supported by natural toxinsand a synthetic analogue to find out if it may help inunderstanding the molecular mechanisms involved inthis peptide–protein interaction.     

Molecular properties of voltage-gated K+ channels

Subfamilies of voltage-activated K⁺ channels (Kv1-4) contribute to controlling neuron excitability and the underlying functional parameters. Genes encoding the multiple subunits from each of these protein groups have been cloned, expressed and the resultant distinct K⁺ currents characterized. The predicted amino acid sequences showed that each subunit contains six putative membrane-spanning -helical segments (S1-6), with one (S4) being deemed responsible for the channels' voltage sensing. Additionally, there is an H5 region, of incompletely defined structure, that traverses the membrane and forms the ion pore; residues therein responsible for K⁺ selectivity have been identified. Susceptibility of certain K⁺ currents produced by the Shaker-related subfamily (Kv1) to inhibition by -dendrotoxin has allowed purification of authentic K⁺ channels from mammalian brain. These are large (M_r 400 kD), octomeric sialoglycoproteins composed of and subunits in a stoichiometry of ()₄()₄, with subtypes being created by combinations of subunit isoforms. Subsequent cloning of the genes for ₁, ₂ and ₃ subunits revealed novel sequences for these hydrophilic proteins that are postulated to be associated with the subunits on the inner side of the membrane. Coexpression of ₁ and Kv1.4 subunits demonstrated that this auxiliary protein accelerates the inactivation of the K⁺ current, a striking effect mediated by an N-terminal moiety. Models are presented that indicate the functional domains pinpointed in the channel proteins.     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

The effect of mechanical load on integrin subunits α5 and β1 in chondrocytes from mature and immature cartilage explants

Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits 5 and 1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits 5 and 1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit 5 in immature and mature cartilage but not in the integrin subunit 1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin 51 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.This work was supported by NIH grant AR45748 to PAT and the HSS MacArthur Cartilage Fund.     

Molecular cloning and heterologous expression in E. coli of cytochrome P45017alpha. Comparison of structural and functional properties of substrate-specific cytochromes P450 from different species

To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017 from species of the Bovidae family (sheep, goat, and bison), which catalyze 17-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C₁₉-steroids. Recombinant cytochromes P45017 were expressed in E. coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography. Highly purified cytochromes P45017 were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC. It is shown that each form of cytochrome P45017 is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b5 in the reaction mixture. The analysis of the activity of the known forms of cytochrome P45017 in view of the data obtained in the present work allows the division of known cytochromes P45017 into three main group: group A (pig, hamster, rat), cytochromes P45017 catalyze the reaction of 17-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17-hydroxyprogesterone and 17-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017, which have no or have insignificant 17,20-lyase activity in relation to 17-hydroxyprogesterone; group C (guinea pig), cytochrome P45017 which either has no or has insignificant 17,20-lyase activity on transformation 17-hydroxypregnenolone to dehydroepiandrosterone.     

Functional Coupling of G Proteins to Endothelin Receptors Is Ligand and Receptor Subtype Specific

1. The aims of the present study were (a) to determine the identity of the G proteins with which the endothelin receptor interacts and whether this interaction is subtype specific and (b) to determine whether agonist exposure can result in specific coupling between the endothelin receptor and G proteins.2. Coupling between endothelin A (ET_A) or endothelin B (ET_B) receptors and G proteins was assessed in two fibroblast cell lines, each expressing one receptor subtype. Four ligands, ET-1, ET-3, SRTXb, and SRTXc, were used for receptor stimulation. The G protein -subunit coupled to the receptor was identified by immunoprecipitation with an antibody against the endothelin receptor and immunoblotting with specific antibodies against different G protein -subunits.3. Unstimulated ET_A and ET_B receptors (ET_AR and ET_BR, respectively) were barely coupled to G_o. The unstimulated ET_AR coimmunoprecipitated with G_i3, whereas the unstimulated ET_BR was much less strongly coupled to G_i3. The coupling of ET_BR to G_i1G_i2 -subunits was much stronger than the coupling of ET_AR to these -subunits. Stimulation with the different ET agonists also resulted in differential coupling of G proteins to the receptor subtypes. All four ligands caused a strong increase in coupling of the ET_BR to G_i3, whereas coupling of the ET_AR to this subunit was not affected by ET-1 and was even decreased by SRTXc. On the other hand, all four ligands caused a much greater increase in the coupling of ET_AR to G_q/G₁₁ than in the coupling of ET_BR to these -subunits. Ligand-induced coupling between the receptors and the G_i1 and G_i2 -subunits is similar for the two receptor subtypes. The same was true for ligand-induced coupling of the receptors to G_o, except that ET-3 increased the coupling of this -subunit to ET_BR and decreased the coupling to ET_AR. Taken together, the results of this study show that coupling between ET receptors and G proteins is ligand and receptor subtype specific.4. It remains to be established whether this diversity of receptor–G protein coupling is of relevance for the various endothelin signaling pathways and/or pathological states.     

Properties,Expression and Potential Roles of Cardiac K<Superscript>+</Superscript> Channel Accessory Subunits: MinK,MiRPs, KChIP,and KChAP

Over the past 10 years, cDNAs encoding a wide range of pore-forming K⁺-channel -subunits have been cloned and found to result in currents with many properties of endogenous cardiac K⁺ channels upon homomeric expression in heterologous systems. However, a variety of remaining discrepancies have led to a search for other subunits that might be involved in the formation of native channels. Over the past few years, a series of accessory subunits has been discovered that modify current properties upon coexpression with -subunits. One of these, the minimal K⁺-channel subunit minK, is essential for formation of the cardiac slow delayed-rectifier K⁺ current, I _Ks, and may also interact in functionally important ways with other -subunits. Another, the K⁺-channel interacting protein KChIP appears critical in formation of native transient outward current (I _to) channels. The roles of 2 other accessory subunits, the minK-related peptide MiRP and the K⁺-channel accessory protein, KChAP, remain unclear. This article reviews the available knowledge regarding the accessory subunits minK, MiRP, KChIP and KChAP, dealing with their structure, effects on currents carried by coexpressed -subunits, expression in cardiac tissues and potential physiological function. On the basis of the available information, we attempt to assess the potential involvement of these accessory K⁺-channel subunits in cardiac pathophysiology and in developing new therapeutic approaches.     

cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca²⁺ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac ₁ and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the ₁ subunit, resulted in Ca²⁺ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the ₁ subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca²⁺ channel functions suggesting that cardiac Ca²⁺ channels expressed in these cells behave, in terms of lack of PKA control, like Ca²⁺ channels of smooth muscle cells.     

Characterization of the G protein coupling of a glucagon receptor to the KATP channel in insulin-secreting cells

The G-protein-mediated coupling of a glucagon receptor to ATP-dependent K channels—K_ATP—has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches, K_ATP channel activity was inhibited by low concentrations of glucagon (IC₅₀ = 2.4 nm); the inhibitory effect vanished at concentrations greater than 50 nm. In cell-attached patches, inhibition by bath-applied glucagon was seen most often, although stimulation was observed in a few cases. A dual action of the hormone is proposed to resolve these apparently divergent results. In excised inside-out patches, K_ATP channel activity was inhibited by addition of subunits purified from either erythrocyte or retina (IC₅₀ = 50 pm and 1 nm, respectively). Subsequent exposure of the patch to _i or _o reversed this effect. In excised inside-out patches, increasing Mg²⁺ in the bath stimulated the channel activity between 0 and 0.5 mm, but blocked it at higher concentrations (IC₅₀ = 2.55 mm). In most cases (70%), GTP had a stimulatory effect at concentrations up to 100 m. However, in three cases, similar GTP levels had clear inhibitory effects. In excised inside-out patches, cholera toxin (CTX) caused channel inhibition. Although the effect could not be reversed by removal of the toxin, the activity was restored by subsequent addition of purified _i or _o . These results are compatible with a model whereby channel inhibition by activated G _S -coupled receptors occurs, at least in part, via association of the subunits of G _S with _i / _o subunits and deactivation of the _i / _o -dependent stimulatory pathway. On the basis of this hypothesis, a model is developed to describe the effects of G proteins on the K_ATP channel, as well as to account for the concentration-dependent stimulation and inhibition of K_ATP channel by Mg²⁺. An interpretation of the ability of glucagon to potentiate, but not initiate, insulin release is also given in terms of this model and the effects of ATP on K_ATP channels.This work was supported by grant DCB-89 19368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R.The authors would like to thank Dr. A.E. Boyd, III for supplying the RINm5F and HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the G protein and subunits from erythrocyte, Dr. R.A. Cerione for supplying the G protein subunit from retina, and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.     

Fibroblast growth factor increases TNFα-mediated prostaglandin E2 production and TNFα receptor expression in human fibroblasts

To examine the possible role of basic fibroblast growth factor (FGF) in regulating the effects of TNF, we tested the effect of FGF on TNF-mediated PGE₂ production and TNF receptor expression in human fibroblasts. We found that, while FGF alone had no effect on PGE₂ production, it enhanced the amount of PGE₂ produced in response to TNF between 3 and 11-fold. FGF stimulated TNF-induced PGE₂ production independent of potential TNF-mediated IL-1 production, as neither anti-IL-1 mAbs nor IL-1 receptor antagonist protein (IRAP) inhibited TNF induced-PGE₂ production or the stimulatory effect of FGF. A one minute exposure of cells to FGF prior to removal was sufficient to significantly enhance TNF-induced PGE₂ production; the maximal FGF effect was reached after a 6 h preincubation. We also found that FGF significantly enhanced TNF receptor expression. Untreated fibroblasts expressed 3,900 receptors/cell, while cells treated with FGF for 6h expressed 9,500 receptors/cell, a 2.4-fold increase in receptor number; there was no apparent change in affinity for TNF (K_d 3.8×10^–11 M). The FGF-mediated increase in TNF receptor expression and TNF-mediated PGE₂ production could be abolished by FGF mAbs, indicating a specific FGF effect. These results show that FGF increases TNF receptor expression and suggest that this may account, at least in part, for the ability of FGF to enhance TNF-mediated PGE₂ production in human fibroblasts.     

GABA A -receptors: Structural requirements and sites of gene expression in mammalian brain

GABA_A-receptors, the major synaptic targets for the neutotransmitter GABA, are gated chloride channels. By their allosteric drug-induced modulation they serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension and epileptiform activity can be regulated. Despite their functional prominence, the structural requirements of fully functional GABA_A-receptors are still elusive. Expression of cDNAs coding for the ₁- and ₁-subunits of rat brain yielded GABA-gated chloride channels which were modulated by barbiturates but displayed only agonistic responses to ligands of the benzodiazepine receptor. GABA_A-receptors with fully functional benzodiazepine receptor sites were formed when the ₁- and ₁-subunits were coexpressed with the ₂-subunit of rat brain. These receptors, however, failed to show cooperativity of GABA in gating the channel. In order to determine the subunit repertoire available for receptor assembly in different neuronal populations in vivo, the sites of subunit gene expression were (₁, ₂, ₃, ₅, ₆, ₁, ₂, ₃, ₂) mapped by in situ hybridization histochemistry in brain sections. The mRNAs of the ₁-, ₁- and ₂-subunits were co-localized e.g. in mitral cells of olfactory bulb, pyramidal cells of hippocampus as well as granule cells of dentate gyrus and cerebellum. The lack of colocalization in various other brain areas points to an extensive receptor heterogeneity. The presence of multiple GABA_A-receptors in brain may contribute to synaptic plasticity, differential responsiveness of neurons to GABA and to variations in drug profiles.Special issue dedicated to Dr. Erminio Costa     

Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences

Voltage-dependent potassium (K_v) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K_v channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K⁺ currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K_v1.3/K_v1.5 channels, which generate the main K_v in macrophages. An increase in K⁺ current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K_v1.3 subunits in the K_v1.3/K_v1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K_v1.3 expression. Neither of these treatments apparently altered the expression of K_v1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K_v1.3/K_v1.5 hybrid channels by altering the subunit stoichiometry.     

Characterization of the G protein coupling of SRIF and β-adrenergic receptors to the maxi KCa channel in insulin-secreting cells

Modulation of the Ca- and voltage-dependent K channel—K_Ca—by receptors coupled to the G proteins G _i /G _o and G _s has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the K_Ca channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous subunits of either G _i or G _o -type G proteins also inhibited the K_Ca channel (IC₅₀ 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses K_Ca channel activity via a membrane-delimited pathway that involves the subunits of PTX-sensitive G proteins G _i and/or G _o . In outside-out patches, activation of G _s either by -agonists or with cholera toxin (CTX) increased K_Ca channel activity, consistent with a membrane-delimited stimulatory pathway linking the -adrenergic receptor to the K_Ca channel via G _s . In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of -agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous _i or _o . Taken together these data suggest that K_Ca channel activity is enhanced by activation of G _s and blocked by activated G _i and/or G _o . Further, K_Ca channel stimulation by activated G _s may be direct, while inhibition by G _i /G _o may involve deactivation of G _s . In inside-out patches K_Ca channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the K_Ca channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous _i or _o . These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G _i /G _o -induced deactivation of G _s .The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G _i and G _o , and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E.     

Cytochemical and immunocytochemical localization of Na,K-ATPase α subunit isoenzymes in the rat heart

In order to understand the functional significance of Na,K-ATPase subunits as well as their isoenzymes, a precise subcellular localization of these in the myocyte is a crucial prerequisite. Cytochemical, immunofluorescence, preembedding immunogold and horse radish peroxidasediaminobenzidine methods, demonstrated ₁ isoenzyme immunoreactivity on the sarcolemma, T-tubules and the subsarcolemmal cisterns of the adult cardiac myocytes. Cytochemically, ouabain resistant Na,K-ATPase precipitate was localized only in the subsarcolemmal cisterns and junctional sarcoplasmic reticulum. For ₂ isoenzyme, immunoreactivity was demonstrated on the sarcolemma as well as in all areas of the myocytes in particularly a close proximation to the sarcoplasmic reticulum and microsomes. For ₃ isoenzyme, only a weak insignificant signal was noted on the sarcolemma, intercalated disc and sarcoplasm. It is suggested that cytochemical ouabain resistant precipitate present in subsarcolemmal cisterns and junctional sarcoplasmic reticulum represent ₁ isoenzyme of Na,K-ATPase. A differential as well as unique localization of subunit isoenzymes of Na,K-ATPase in specific structures of cardiac myocytes may suggest importance in physiological function at these sites.     

Modulation of the inwardly rectifying K+ channel in isolated human atrial myocytes by α1-adrenergic stimulation

We have examined the ₁-adrenergic modulation of the inwardly-rectifying K⁺ channel (I _K1) in isolated human atrial myocytes using the patch clamp technique. ₁-Adrenergic agonist methoxamine produced action potential prolongation and a depolarization of the resting membrane potential. Under whole-cell voltage clamp conditions, bath application of methoxamine can inhibit macroscopic I _K1. The methoxamine-induced inhibition was reversible and concentration dependent, with the concentration for half-maximal inhibition being 18 m. The methoxamine-induced inhibition of I _K1 was prevented by bath application of ₁-adrenergic blocker prazosin. The current was similarly inhibited by phorbol ester (PMA), an activator of protein kinase C (PKC). In contrast, methoxamine failed to inhibit the current in the presence of a specific PKC inhibitor H-9, suggesting that PKC is involved in the methoxamine-induced inhibition of I _K1. In single channel recording from cell attached patches, bath-applied methoxamine could suppress I _K1 channels by decreasing the frequency and duration of bursting without affecting unitary amplitude. Direct application of purified PKC to excised inside-out patches inhibited channel activity similar to methoxamine in cell-attached patches. The PKC selective inhibitor, PKC19-36, prevented the PKC-induced inhibition of the channel. We conclude that human atrial I _K1 can be inhibited by ₁-adrenergic stimulation via PKC-dependent pathways.     

Control of K+ channels by G proteins

Heterotrimeic G proteins are thought to couple receptors to ionic channels via cytoplasmic mediators such as cGMP in the case of retinal rods, cAMP in the case of olfactory cells, and the cAMP cascade in the case of cardiac myocytes. G protein-mediated second messenger effects on K⁺ channels are dealt with elsewhere in this series. Recently, membrane-delimited pathways have been uncovered and an hypothesis proposed in which the subunits of G proteins directly couple receptors to ionic channels, particularly K⁺ channels. While direct coupling has not been proven, the membrane-delimited nature has been established for specific G proteins and their specific K⁺ channel effectors.